Project Summary The World Health Organization cites multi-drug resistant (MDR) bacteria as one of the top three threats to human health. Unfortunately, antibiotic development has essentially ground to a halt thanks to these rapidly emerging resistant strains. No one wants to invest in a therapeutic with such a short shelf life. The need for a viable, broad treatment strategy is overwhelming. The only new classes of antibiotics introduced in the last twenty years are active solely against Gram-positive bacteria and resistance to these is already endemic. And that's the good news. Much worse, there have been no real advances in treating Gram- negative infections whatsoever. Clinicians have adopted the following mantra: for the treatment of Gram- positives we need better drugs; for Gram-negatives we need any drugs. Without a deeper understanding of the bacterial resistance/persistence and transmission mechanisms, along with the development of new approaches to combat these MDR pathogens, countless people will die. Our studies have two aims. First, we would like to better understand the protective mechanisms that bacteria employ to overcome antibiotics and the host immune response. Second, we would like to impede these mechanisms using small molecule intervention. During the last funding period we elucidated functional processes and molecular recognition characteristics of proteins involved in a variety of bacterial responses, including biofilm formation and environmental sensing. These studies were aimed at identifying 'druggable' points in signal transduction pathways. As a result of these efforts, we were able to develop an arsenal of compounds (referred to as 2AIs) that re-sensitize MDR bacteria (both Gram-positive and Gram-negative) to current antibiotics. We have demonstrated the efficacy of these compounds against 20-plus bacterial strains. Here we will characterize a complete signaling pathway responsible for biofilm matrix production in order to determine sites for possible therapeutic intervention, elucidate the mechanism of action of our 2AI compounds, develop improved 2AI compounds and evaluate lead 2AIs to act as adjuvants to current antibiotics in an animal model.